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H4R) and the Human Free Fatty Acid Receptor 4 (FFA4)

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H4R) and the Human Free Fatty Acid Receptor 4 (FFA4) Evaluation of signalling and phosphorylation responses of the human histamine H4 receptor (H4R) and the human free fatty acid receptor 4 (FFA4) Thesis Submitted for the Degree of Doctor of Philosophy at the University of Leicester By Sajjad Ali Riaz Department of Molecular and Cellular Biology University of Leicester May 2016 Abstract The knowledge that G protein-coupled receptors (GPCRs) are regulated by phosphorylation in a process that results in the recruitment of arrestins, leading to receptor desensitisation is now well known. The histamine H4 receptor (H4R) and the free fatty acid receptor 4 (FFA4) are family A GPCRs that both have the ability to become phosphorylated in their third intracellular loops and C-terminal tails by kinases found in the cytosolic milieu of the cells and tissues they are expressed in. Investigations into the phosphorylation status of the histamine H4 receptor have revealed a receptor that is highly phosphorylated even in the basal state. The endogenous, full agonist for the human H4R, histamine, induced a robust increase in receptor phosphorylation. However, the β-arrestin-biased agonist JNJ7777120 did not. Extending this study using mass spectrometry revealed the individual sites of phosphorylation. Histamine and JNJ7777120 also caused H4R internalisation. Our data suggests a similar level of endocytosis induced by histamine and JNJ7777120 at 5 or 30 min stimulation. Thus, we show that JNJ7777120, while previously demonstrating its differing effects on H4R signalling, also shows differences in the phosphorylation of the H4R when compared to histamine. Using the wild type FFA4 receptor and its phosphorylation-deficient mutants, we show the importance of phosphorylation in the recruitment of arrestin to the receptor as well as delineating G protein-dependent and independent downstream signalling pathways. Knowledge of the different signalling cascades and their mechanism of activation would be useful in the design of biased ligands for therapeutic benefits in order to develop safer and more efficacious drugs. The use of a FFA4 receptor which is phosphorylation-deficient and, therefore, couples to arrestin-3 in a reduced manner may be useful in proof-of-concept studies where the downstream signalling in a physiological setting is mediated by arrestin as opposed to G proteins. Further evidence of the importance of phosphorylation is provided by my work with the phosphomimetic FFA4 mutant receptor, which I show does not faithfully mimic phosphorylated serine and/or threonine residues resulting, in a reduced ability to couple to arrestin-3. ii Publications Papers Bradley, S.J., Riaz, S.A., and Tobin, A.B. (2014) Employing novel animal models in the design of clinically efficacious GPCR ligands. Curr Opin Cell Biol. 27 (100): 117- 25 Abstracts Riaz, S.A., Charlton, S.J., and Tobin, A.B. Regulation of the M1 RASSL by Phosphorylation. 4th Focused Meeting Cell Signalling 23rd-24th April 2012. Poster Presentation. Riaz, S.A., Charlton, S.J., Butcher, A.J., and Tobin, A.B. Studies to Validate a Chemical Genetics Approach to Investigate the M1 Muscarinic Acetylcholine Receptor. 13th Annual Joint meeting of the Great Lakes GPCR Retreat 17th-19th October 2012. Poster Presentation. iii Acknowledgements “Verily, with every hardship comes ease” First, and foremost, all praise is due to Allah, the Lord of the worlds. The Beneficent, the Merciful. I would like to extend a big thank you to my supervisor Prof. Andrew Tobin for his continued support, encouragement, and contributions throughout my PhD. My PhD was funded by the BBSRC and Novartis. Thank you to my industrial supervisor Prof. Steven Charlton. I would also like to acknowledge my committee members Prof. John Challiss and Dr. Gary Willars for their valuable contributions and feedback. Thank you to all the members of Lab 202 both past and present. To Mahmood for the squash matches and the attempts to make me do “just one post-doc”, but most importantly for feeding me during every Ramadan and being a brother from another mother. To my fellow PhD students in the lab Omar, Andy, and Simon…stay strong guys! To Lev and Debbie. To Jules who routinely put up with old and/or French jokes aimed in her direction. To Strongo for the Spanish lessons and spotting me at the gym. To Rudi for his countless conversations regarding fantasy football tactics that were shaky at best. And to Tim, who made me realise that no matter what, at least my jokes weren’t as bad as his. To Sophington, who has truly become a friend for life and has made the last couple of years awesome. For providing a listening ear to my incessant complaining and a shoulder to cry on during my incessant weeping. Please note, this does not mean I’d go easy on you if ever you pushed me too far. I extend the greatest thanks and appreciation to Adrian. Who has been with me through all the highs and the many, many lows of this PhD during the past four and a bit years. Whose feedback and help with every experiment has been invaluable, and who was so generous in letting me piggy back on his FFA4 project. I hope I can count you as another friend for life. To the friends and family away from the lab. To Kamal “Newsnight” Chana and Jasminder “Halo Fetish” Sahota, two of the best housemates one could ask for…and two of the worst gym partners. Finally, a thank you to my family for still not knowing what I actually do and yet being there with me through it all. To the best brother and sister ever for your love and support, for acknowledging I am the favourite child, and for bestowing upon me the title “Golden Boy”. To my beloved parents who are the sun that brightens my day, the noor of my eyes, and the greatest source of my strength. Your unconditional love, support, and sacrifices have made me the man I am today. I am forever grateful to you and love you both immeasurably. Thank you for your belief in me, I just hope I have made you proud. This thesis is dedicated to Ba and Ama, who passed away in the first and last years of my PhD. May Allah grant you the highest place in Jannah, ameen. iv Table of Contents Abstract ............................................................................................................................. ii Publications ...................................................................................................................... iii Acknowledgements .......................................................................................................... iv Table of Contents .............................................................................................................. v Abbreviations ................................................................................................................... ix Chapter 1 | Introduction .................................................................................................... 1 1.1. G protein-coupled receptors ............................................................................... 1 1.1.1. Classification of GPCRs ............................................................................. 1 1.1.2. GPCR activation ......................................................................................... 5 1.1.3. G-protein dependent signalling ................................................................... 6 1.1.4. G-protein independent signalling .............................................................. 10 1.1.5. GPCR regulation by phosphorylation ....................................................... 12 1.1.6. Biased agonism or functional selectivity .................................................. 15 1.2. Histamine receptors .......................................................................................... 16 1.2.1. Classification and alternative splicing of the histamine receptors ............ 16 1.2.2. Localisation of the histamine receptors .................................................... 17 1.2.3. Structural architecture of histamine receptors .......................................... 21 1.2.4. Downstream signalling of histamine receptors ......................................... 27 1.2.5. Function of histamine receptors ................................................................ 30 1.3. Free fatty acid receptors ................................................................................... 33 1.3.1. Localisation and physiological function of the free fatty acid receptors .. 34 1.3.2. Structural architecture of free fatty acid receptors .................................... 38 1.3.3. Downstream signalling of free fatty acid receptors .................................. 40 1.4. Thesis aims ....................................................................................................... 43 Chapter 2 | Materials and Methods ................................................................................. 44 2.1. Materials ........................................................................................................... 44 2.1.1. Standard reagents, chemicals and consumables ....................................... 44 2.1.2. Specific reagents and assay kits ................................................................ 44 2.1.3. Bacterial strains ......................................................................................... 45 2.1.4. DNA plasmids and receptor constructs .................................................... 45 2.1.5. Mammalian cell lines ...............................................................................
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